Peptide capable for binding to iridium

ABSTRACT

The present invention provides a novel peptide capable for binding to iridium. The peptide consists of an amino acid sequence represented by SQMMGHMGHGNMNHMNHGGKFDFHH.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of Japanese Patent Application No.:2012-089787 filed Apr. 11, 2012, the content of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION Technical Field

The present invention relates to a peptide capable for binding toiridium.

SUMMARY Technical Problem

The purpose of the present invention is to provide a novel peptidecapable for binding to iridium.

Solution to Problem

The present invention is a peptide capable for binding iridium, wherein

the peptide consists of an amino acid sequence represented by SEQ ID NO:01.

The present invention is a method for binding a peptide to iridium, themethod comprising steps of:

mixing the iridium with a solution containing the peptide consisting ofan amino acid sequence represented by SEQ ID NO: 01 so as to bind thepeptide represented by an amino acid sequence represented by SEQ ID NO:01 to the iridium.

Advantageous Effects of Invention

The present invention provides a novel peptide capable for binding toiridium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic view of a two-component composite 103 of avidin101 and biotin-PEG12 (reference number: 102) according to thecomparative example 1, which is included in the experiment 1.

FIG. 2 shows a schematic view of a composite 106 of avidin 104 andbiotin-PEG12-peptide 105 according to the example 1, which is includedin the experiment 1.

FIG. 3 shows a schematic view of a three-component composite 108 ofiridium nanoparticle 107, avidin 104, and biotin-PEG12-peptide 105according to the example 1, which is included in the experiment 1.

FIG. 4 is an electrophoretic photograph showing the results of theexperiment 1.

DETAILED DESCRIPTION

The embodiment of the present invention is described below.

The present inventor has discovered that the peptide consisting of theamino acid sequence represented by SQMMGHMGHGNMNHMNHGGKFDFHH (SEQ ID NO:01) recognizes iridium. The present invention is provided on the basisof this discovery.

The peptide consisting of the amino acid sequence represented by SEQ IDNO: 01 is derived from the amino acid sequence of the 382^(nd) to the406^(th) amino acids of CueO. The term “CueO” means a multi-copperoxidase of Escherichia coli K-12 strain.

(Experiment)

The following experiments describe the present invention in more detail.

(Experiment 1)

The experiment 1 is comprised of the example 1 and the comparativeexample 1.

In the experiment 1, a pull-down method was performed. The pull-downmethod according to the experiment 1 is easily described below.

In the pull-down method according to the experiment 1, three-componentcomposite of avidin, biotin-PEG12-peptide, and a iridium nanoparticle isformed in an aqueous solution. After the aqueous solution containing thethree-component composite is subjected to a centrifugal separation,three-component composite are not collected as a supernatant. In otherwords, three-component composite is left in the aqueous solution as aprecipitation (i.e., residue). Subsequently, the three-componentcomposite is decomposed by heat treatment, and the solution is subjectedto an electrophoresis. After the electrophoresis, a band of the subunitmonomer of the avidin is observed.

On the other hand, two-component composite of avidin and biotin-PEG12 isnot bound to a iridium nanoparticle. After an aqueous solutioncontaining the two-component composite is subjected to a centrifugalseparation, the two-component composite is eliminated as a supernatant.After the aqueous solution is heated, the aqueous solution is subjectedto an electrophoresis. However, since the avidin has been eliminated asone chemical component contained in the supernatant, a band of thesubunit monomer of the avidin is not observed.

In the experiment 1, the following solutions were prepared.

Biotin-PEG12-peptide solution

Biotin-PEG12 solution

Tris hydrochloric acid buffer solution

Avidin aqueous solution

Two-component composite aqueous solution of avidin and biotin-PEG12 (SeeFIG. 1)

Composite aqueous solution of avidin and biotin-PEG12-peptide (See FIG.2)

Polyoxyethylene (20) sorbitan monolaurate aqueous solution

Buffer solution containing polyoxyethylene (20) sorbitan monolaurate

Sodium dodecylsulfate solution

Glycerol solution

Aqueous solution for an electrophoresis using a denaturingpolyacrylamide gel

Iridium nanoparticle dispersion liquid

A detailed method for preparing these chemical reagents is describedbelow.

(Preparation of a Biotin-PEG12-Peptide Solution)

The present inventor requested synthesis of a compound represented bythe following formula (I) (hereinafter, referred to as“biotin-PEG12-peptide”) from Sigma Aldrich Japan Co., Ltd. A solid-phasesynthesis method was used for the synthesis of the biotin-PEG12-peptide.

(Chem 01) (SEQ ID NO: 1) Biotin-PEG12-SQMMGHMGHGNMNHMNHGGKFDFHH (I)

The biotin-PEG12-peptide prepared by Sigma Aldrich Japan Co., Ltd. had apurity of 96.2%.

In the biotin-PEG12-peptide, as is clear from the formula (I), theN-terminal of the peptide consisting of the amino acid sequencerepresented by SEQ ID NO: 01 is chemically modified withbiotin-polyethylene glycol.

The method for modifying the N-terminal of the peptide withbiotin-polyethylene glycol is well known. As one example, EZ-LinkNHS-PEG12-biotin (available from Thermo Fisher Scientific Corporation)may be used.

The EZ-Link NHS-PEG12-biotin has the following chemical formula (II).

The term “NHS” means N-hydroxysuccinimide. NHS binds to an active estergroup. When a peptide is added, the ester group activated by NHS reactsimmediately with an NH₂ group located at the N terminal of the peptideor the side-chain of a lysine residue to form an amide bond.

The term “PEG” means polyethylene glycol. In more detail, PEG isrepresented by the following chemical formula (III).

—(—CH₂—CH₂—O)_(n)—  (III)

where n is a natural number.

It is desirable that the value of n is not less than 1 and not more than20. The term “PEG12” means that the PEG where is n=12.

It is believed that the non-specific interaction between two moleculescoupled through polyethylene glycol is prevented by the polyethyleneglycol. In other words, it is believed that such a polyethylene glycolsuppresses the non-specific interaction between two biological moleculeschemically bonded to the both ends of the polyethylene glycol. This isbecause the two molecules are located far away from each other due tothe polyethylene glycol interposed between the two molecules, as isclear from the chemical formula (II). For more detail, see Non PatentLiterature 1, if necessary.

[Non Patent Literature 1] George et. al., “Effect of Surface Wettabilityon the Adsorption of Proteins and Detergents”, Journal of the AmericanChemical Society, Vol. 120, pp 3464-3473 (1998)

Therefore, in the chemical formula (I), the biotin molecule and thepeptide consisting of the amino acid sequence represented by SEQ ID NO:01 does not interact with each other due to PEG12.

(Preparation of the Biotin-PEG12-Peptide Solution)

Biotin-PEG12-peptide obtained from Sigma Aldrich Japan Co., Ltd. wasdissolved with ultrapure water to prepare a biotin-PEG12-peptidesolution having a concentration of 0.1 mM. The preparedbiotin-PEG12-peptide solution was kept under a temperature of −30degrees Celsius.

(Preparation of the Biotin-PEG12 Solution)

EZ-Link NHS-PEG12-Biotin (available from Thermo Fisher Scientificcompany) was dissolved with dimethylsulfoxide to prepare an EZ-LinkNHS-PEG12-Biotin solution having a concentration of 10 mM.

Then, chemical reagents shown in Table 1 were mixed. The mixture liquidwas left under a temperature of 25 degrees Celsius for one hour.

TABLE 1 Chemical reagent Volume (unit: microliter) 10 mMNHS-PEG12-Biotin 20 1M Tris hydrochloric acid 20 buffer solution (pH: 8)Ultrapure water 160

In this way, the biotin-PEG12 solution having a concentration of 1 mMwas prepared. This biotin-PEG12 solution was diluted with water toprepare the biotin-PEG12 solution having a concentration of 0.1 mM.

(Preparation of the Tris Hydrochloric Acid Buffer Solution)

Hydrochloric acid was added in Tris buffer solutions to prepare thefollowing four kinds of Tris hydrochloric acid buffer solutions shown inTable 2.

TABLE 2 1M Tris hydrochloric acid buffer solution (pH: 8) 100 mM Trishydrochloric acid buffer solution (pH: 8)  10 mM Tris hydrochloric acidbuffer solution (pH: 8) 1M Tris hydrochloric acid buffer solution (pH:6.8)

(Preparation of the Avidin Aqueous Solution)

Avidin (Available from Thermo Fisher Scientific company, trade name:NeutrAvidin, 10 milligrams) was dissolved with ultrapure water toprepare 10 milligram/milliliter of an avidin aqueous solution. In FIG.1, the avidin has a reference sign 101.

(Preparation of Two-Component Composite of Avidin and Biotin-PEG12)

The chemical reagents shown in the following Table 3 were mixed. In FIG.1, the biotin-PEG12 has a reference sign 102. The mixture was left undera temperature of 23 degrees Celsius for two hours.

TABLE 3 Volume Chemical reagent (unit: microliter) 10 mg/mL of avidin 501M Tris hydrochloric acid 5 buffer solution (pH: 8) Ultrapure water 950.1 mM biotin-PEG12 350

Subsequently, the mixture (500 microliters) was filtrated using acentrifugal ultrafiltration unit (available from Merck Ltd., trade name:Amicon Ultra-0.5, MWCO: 30,000) to obtain a residue. The centrifugalseparation was performed under a temperature of 4 degrees Celsius at anrpm of 10,000×g for 20 minutes. In this way, the mixture wasconcentrated to obtain a concentrated liquid of approximately 50microliters.

A Tris hydrochloric acid buffer solution (pH: 8, 450 microliters) havinga concentration of 10 mM were mixed to the concentrated liquid. Theconcentrated liquid was subjected to a centrifugal separation under thesame conditions as above again to obtain a concentrated liquid ofapproximately 50 microliters again. This was repeated once again.

The concentrated liquid was diluted with the Tris hydrochloric acidbuffer solution (pH: 8) having a concentration of 10 mM to obtain anaqueous solution containing a two-component composite of avidin andbiotin-PEG12. This aqueous solution had a volume of 500 microliter. InFIG. 1, two-component composite of avidin and biotin-PEG12 have areference sign 103.

(Preparation of Composite of Avidin and Biotin-PEG12-Peptide)

The procedure similarly to that of the preparation of two-componentcomposite of avidin and biotin-PEG12 was conducted, except that thechemical reagents shown in the following Table 4 were mixed instead ofthe chemical reagents shown in Table 3, so as to prepare an aqueoussolution containing a composite of avidin and biotin-PEG12-peptide.

TABLE 4 Volume Chemical reagent (unit: microliter) 10 mg/mL of avidin 501M Tris hydrochloric acid buffer solution (pH: 8) 5 Ultrapure water 950.1 mM biotin-PEG12-peptide (See the experiment 1) 350

In FIG. 2, the avidin has a reference sign 104. The biotin-PEG12-peptidehas a reference number 105. The composite has a reference number 106.

(Preparation of Polyoxyethylene (20) Sorbitan Monolaurate AqueousSolutions)

An aqueous solution of polyoxyethylene (20) sorbitan monolaurate(available from Affymetrix company, trade name: ANAPOE-20) having aconcentration of 10 percent by weight was diluted with ultrapure waterto prepare an aqueous solution of polyoxyethylene (20) sorbitanmonolaurate having a concentration of 1 percent by weight. Similarly, anaqueous solution of polyoxyethylene (20) sorbitan monolaurate having aconcentration of 0.1 percent by weight was prepared. Polyoxyethylene(20) sorbitan monolaurate may be used as a surfactant.

(Preparation of the Buffer Solution Containing Polyoxyethylene (20)Sorbitan Monolaurate)

The two kinds of buffer solutions shown in Table 5 were prepared.

TABLE 5 Buffer solution 10 mM Tris hydrochloric acid buffer solution(pH: 8) containing 0.1% by weight polyoxyethylene (20) sorbitanmonolaurate 10 mM Tris hydrochloric acid buffer solution (pH: 8)containing 0.2% by weight polyoxyethylene (20) sorbitan monolaurate

(Preparation of the Sodium Dodecyl Sulfate Aqueous Solution)

Sodium dodecyl sulfate (available from Wako Pure Chemical IndustriesLtd.) was dissolved with ultrapure water to prepare a sodium dodecylsulfate aqueous solution having a concentration of 10% by weight.

(Preparation of the Glycerol Aqueous Solution)

Glycerol (available from Life Technologies Corporation) was dissolvedwith ultrapure water to prepare a glycerol aqueous solution having aconcentration of 50% by weight.

(Preparation of an Aqueous Solution for an Electrophoresis Using aDenaturing Polyacrylamide Gel)

The chemical reagents shown in the following Table 6 were mixed toprepare an aqueous solution. Furthermore, approximately 1 milligram ofbromophenol blue (powder) was added to the aqueous solution.Subsequently, the aqueous solution was stirred well. In this way,prepared was the aqueous solution for an electrophoresis using adenaturing polyacrylamide gel.

TABLE 6 Solution Volume 1M Tris hydrochloric acid buffer solution (pH:6.8) 0.55 milliliters 50% by weight glycerol solution 3.6 milliliters2-mercaptoethanol 0.5 milliliters 10% by weight sodium dodecyl sulfate 4milliliters Ultrapure water 1.35 milliliters

(Preparation of the Iridium Nanoparticle Dispersion)

A dispersion liquid of the iridium nanoparticles each having a diameterof 1-4 nanometers (available from Renaissance Energy Research company,0.5 milliliters) was mixed with ultrapure water (0.5 milliliters).

The mixture was subjected to a centrifugal separation under atemperature of 4 degrees Celsius at an rpm of 290,000×g for 2 hours.Subsequently, the supernatant was eliminated to obtain the concentratedliquid having a volume of 100 microliters. In this way, the mixture wasconcentrated.

Then, the 0.1% by weight polyoxyethylene (20) sorbitan monolaurateaqueous solution was added to the concentrated liquid. Subsequently, theconcentrated liquid was subjected to a centrifugal separation againunder the above-mentioned condition to remove the supernatant. In thisway, a concentrated liquid having a volume of 100 microliters wasobtained.

Again, the 0.1% by weight polyoxyethylene (20) sorbitan monolaurate wasadded to the concentrated liquid. Subsequently, the concentrated liquidwas subjected to a centrifugal separation again under theabove-mentioned condition to remove the supernatant. In this way, aconcentrated liquid having a volume of 100 microliters was obtainedagain.

A 0.1% by weight polyoxyethylene (20) sorbitan monolaurate aqueoussolution was added to the concentrated liquid to obtain the iridiumnanoparticle dispersion liquid having a volume of 0.1 milliliter.

EXAMPLE 1

The chemical reagents shown in the following Table 7 were mixed. Themixture was left under a temperature of 25 degrees Celsius for tenminutes.

TABLE 7 Volume Chemical reagent (unit: microliter) 10 mM Trishydrochloric acid buffer solution (pH: 8) 24 10 mM Tris hydrochloricacid buffer solution (pH: 8) 25 containing 0.2% by weight ofpolyoxyethylene (20) sorbitan monolaurate Iridium nanoparticledispersion 50 Composite aqueous solution of 1 avidin andbiotin-PEG12-peptide

In FIG. 3, the iridium nanoparticles have a reference sign 107. Thecomposite of Avidin and biotin-PEG12-peptide have a reference sign 106.The three-component composite of the iridium nanoparticles, avidin, andbiotin-PEG12-peptide have a reference sign 108. The subunit monomer ofthe avidin obtained by heating the avidin has a reference sign 109. Thebiotin-PEG12-peptide after the heating has a reference sign 110.

The mixture was subjected to a centrifugal separation under atemperature of 4 degrees Celsius at an rpm of 290,000×g for 30 minutes.Subsequently, the supernatant was removed to eliminate the unreactedbiotin-PEG12-peptide. In this way, a liquid mixture was concentrated toobtain a concentrated liquid (i.e., residue) having a volume of 10microliters.

Then, the Tris hydrochloric acid buffer solution (pH: 8, 10 mM, 200microliters) containing 0.1% by weight polyoxyethylene (20) sorbitanmonolaurate was added to the concentrated liquid. Subsequently, theconcentrated liquid was subjected to a centrifugal separation againunder the above-mentioned condition to remove the supernatant. In thisway, a concentrated liquid (i.e., residue) having a volume of 10microliters was obtained.

The Tris hydrochloric acid buffer solution (pH: 8, 10 mM, 200microliters) containing 0.1% by weight polyoxyethylene (20) sorbitanmonolaurate was added to the concentrated liquid again. Subsequently,the concentrated liquid was subjected to a centrifugal separation againunder the above-mentioned condition to remove the supernatant. In thisway, a concentrated liquid (i.e., residue) having a volume of 10microliters was obtained again. This concentrated liquid wasbrownish-red. Since a iridium nanoparticle dispersion liquid isbrownish-red, it was confirmed by the naked eyes that the obtainedconcentrated liquid (i.e., residue) contained iridium nanoparticles.

The aqueous solution for an electrophoresis using a denaturingpolyacrylamide gel was added to the concentrated iridium particledispersion liquid. The volume of the aqueous solution was 20microliters.

Subsequently, an ultrasonic wave was applied to the aqueous solutionusing an ultrasonic wave washer (available from AS ONE Corporation,trade name: USK-1A, configuration: High).

The concentrated iridium nanoparticle dispersion was heated under atemperature of 98 degrees Celsius for ten minutes to decompose theavidin molecule to avidin subunit monomers.

After the heating, the concentrated iridium nanoparticle dispersion wassubjected to a centrifugal separation at an rpm of 10,000×g for oneminute. In this way, a sample solution according to the example 1 wasobtained.

COMPARATIVE EXAMPLE 1

The procedure similarly to that of the example 1 was conducted, exceptthat the biotin-PEG12 was used instead of the biotin-PEG12-peptide. Inthis way, a sample solution according to the comparative example 1 wasobtained.

(Electrophoresis in the Experiment 1)

These two kinds of sample solutions according to the example 1 and thecomparative example 1 were subjected to electrophoresis. The detail ofthe electrophoresis is described below.

A polyacrylamide gel (pre-cast gel, available from Bio-Rad Laboratories,Inc., trade name: 12% Mini-PROTEAN TGX) was equipped to anelectrophoresis tank. As an electrophoretic buffer, a pre-mixed buffer(available from Bio-Rad Laboratories, Inc., trade name: premix buffer10× Tris/glycine/SDS) was diluted ten times with extrapure water. Thisdiluted solution was poured to the electrophoresis tank.

The two sample solutions (for each 15 microliters) according to theexample 1 and the comparative example 1 were added to the wells providedat the top of the polyacrylamide gel.

A benchmark protein ladder (available from Life TechnologiesCorporation, 5 microliters) was added the well provided at the top ofthe polyacrylamide gel as a molecular weight marker.

The electrophoresis was performed under a constant voltage of 150 volts.

When the Bromophenol Blue contained in the aqueous solution was moved bythe electrophoresis to the neighborhood of the bottom of thepolyacrylamide gel, the electrophoresis was stopped. Subsequently, a CBBstain was performed with a CBB stain kit (available from Bio-RadLaboratories, Inc., trade name: R-250 stain & destain kit).

The image of the stained polyacrylamide gel was read with a flatbedscanner (Available from CANON. Inc., trade name: CanoScan D2400U). Theband of the subunit monomer of the avidin was quantified using imageprocessing software ImageJ (available from National Institutes ofHealth) on the basis of the read image.

FIG. 4 shows the electrophoretic results.

As shown in FIG. 4, a clear band was observed in the example 1. On theother hand, such a clear band was not observed in the comparativeexample 1.

According to the image processing software, the band according to theexample 1 contained 3.9 times as many subunit monomers of avidin as theband according to the comparative example 1.

From the electrophoresis result shown in FIG. 4, namely, the result by apull-down method, it was found that Biotin-PEG 12-peptide binds to airidium nanoparticle to form an aggregate (i.e., precipitate). Since thebiotin and the peptide do not interact due to PEG 12, it was found thatthe peptide consisting of the amino acid sequence represented by SEQ IDNO: 01 binds to iridium. In other words, it was found that the peptideconsisting of the amino acid sequence represented by SEQ ID NO: 01recognizes iridium.

INDUSTRIAL APPLICABILITY

The present invention provides a peptide capable for binding to iridium.

REFERENCE SIGNS LIST

-   101, 102: precipitate-   101, 104: avidin-   102: biotin-PEG 12-   103: two-component composite of avidin and biotin-PEG12-   105, 110: biotin-PEG12-peptide-   106: composite of avidin and biotin-PEG12-peptide-   107: iridium nanoparticle-   108: three-component composite of iridium nanoparticle, avidin, and    biotin-PEG 12-peptide-   109: subunit monomer of avidin

1. A peptide capable for binding iridium, wherein the peptide consistsof an amino acid sequence represented by SEQ ID NO:
 01. 2. A method forbinding a peptide to iridium, the method comprising steps of: mixing theiridium with a solution containing the peptide consisting of an aminoacid sequence represented by SEQ ID NO: 01 so as to bind the peptiderepresented by an amino acid sequence represented by SEQ ID NO: 01 tothe iridium.